Mechanistic understanding and inhibition of Zika NS5 protein ABSTRACT Zika virus (ZIKV) belongs to the single-stranded RNA-containing flavivirus family. Its recent outbreak and implication in human diseases (e.g. neurological disorders) have raised a global health alarm, and urgency to develop a therapeutic strategy against ZIKV infection. However, there are no currently approved antivirals against ZIKV available yet. This application seeks to develop an antiviral strategy against the non-structural protein 5 (NS5) of ZIKV, which is responsible for virus-specific genomic replication. On one hand, the currently identified flavivirus inhibitors will be evaluated for their efficiency on ZIKV inhibition. On the other hand, mechanistic details of ZIKV NS5-mediated RNA replication will be investigated, thereby providing a basis for development of synergistic inhibition strategies targeting various enzymatic steps of ZIKV NS5. In Aim 1, structural, biochemical and cellular approaches will be taken to evaluate the inhibition of ZIKV NS5-mediated de novo RNA synthesis by the thiophenyl propargyl alcohol (TPA) compounds, the non-nucleoside inhibitors (NNIs) that have been identified as inhibitors for Dengue virus (DENV) NS5, in vitro and ex vivo. Our recent structural study of ZIKV NS5 revealed that the TPA-binding site of DENV NS5 is conserved in ZIKV NS5. Through evaluation of the inhibitory effects of the TPA compounds on ZIKV NS5, this application will address whether the TPA compounds can serve as inhibitors to ZIKV NS5, and more importantly, to provide a basis for structure-based drug optimization for ZIKV NS5. In Aim 2, the mechanistic basis of ZIKV NS5-mediated RNA replication will be determined through structure elucidation of the replication initiation and elongation complexes of ZIKV NS5, combined with mutational and enzymatic analyses. The structural knowledge on the conformational transition of ZIKV NS5 from replication initiation to elongation will then provide a framework for structure-based drug design for comprehensive inhibition of ZIKV NS5 activity. Together, the proposed studies will provide key mechanistic insights into the NS5-mediated genome replication and establish a foundation for development of effective inhibitors against ZIKV.